Bugnicourt Amandine, Froissard Marine, Sereti Kostianna, Ulrich Helle D, Haguenauer-Tsapis Rosine, Galan Jean-Marc
Institut Jacques Monod-Centre National de la Recherche Scientifique/Universités Paris 6 and 7, 75251 Paris Cedex 05, France.
Mol Biol Cell. 2004 Sep;15(9):4203-14. doi: 10.1091/mbc.e04-05-0420. Epub 2004 Jun 23.
In Saccharomyces cerevisiae, deficiencies in the ESCRT machinery trigger the mistargeting of endocytic and biosynthetic ubiquitinated cargoes to the limiting membrane of the vacuole. Surprisingly, impairment of this machinery also leads to the accumulation of various receptors and transporters at the plasma membrane in both yeast and higher eukaryotes. Using the well-characterized yeast endocytic cargo uracil permease (Fur4p), we show here that the apparent stabilization of the permease at the plasma membrane in ESCRT mutants results from an efficient recycling of the protein. Whereas several proteins as well as internalized dyes are known to be recycled in yeast, little is known about the machinery and molecular mechanisms involved. The SNARE protein Snc1p is the only cargo for which the recycling pathway is well characterized. Unlike Snc1p, endocytosed Fur4p did not pass through the Golgi apparatus en route to the plasma membrane. Although ubiquitination of Fur4p is required for its internalization, deubiquitination is not required for its recycling. In an attempt to identify actors in this new recycling pathway, we found an unexpected phenotype associated with loss of function of the Vps class C complex: cells defective for this complex are impaired for recycling of Fur4p, Snc1p, and the lipophilic dye FM4-64. Genetic analyses indicated that these phenotypes were due to the functioning of the Vps class C complex in trafficking both to and from the late endosomal compartment.
在酿酒酵母中，内体分选转运复合体（ESCRT）机制的缺陷会导致内吞和生物合成途径中泛素化货物错误靶向至液泡的限制膜。令人惊讶的是，该机制的损伤还会导致酵母和高等真核生物中各种受体和转运蛋白在质膜上积累。利用特征明确的酵母内吞货物尿嘧啶通透酶（Fur4p），我们在此表明，ESCRT突变体中通透酶在质膜上的明显稳定是由于该蛋白的有效循环利用。虽然已知酵母中有几种蛋白质以及内化染料会被循环利用，但对于其中涉及的机制和分子机制却知之甚少。SNARE蛋白Snc1p是唯一一种循环途径得到充分表征的货物。与Snc1p不同，内吞的Fur4p在返回质膜的途中不经过高尔基体。虽然Fur4p的泛素化是其内化所必需的，但其循环利用并不需要去泛素化。为了确定这条新的循环途径中的作用因子，我们发现了一种与Vps C类复合体功能丧失相关的意外表型：该复合体缺陷的细胞在Fur4p、Snc1p和亲脂性染料FM4-64的循环利用方面存在缺陷。遗传分析表明，这些表型是由于Vps C类复合体在往返晚期内体区室的运输过程中的功能所致。
